BioMed Central
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Virology Journal
Open Access
Research
Clearance of an immunosuppressive virus from the CNS coincides
with immune reanimation and diversification
Henning Lauterbach
1
, Phi Truong
1
and Dorian B McGavern*
1,2
Address:
1
Molecular and Integrative Neurosciences Department, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037,
USA and
2
Harold L. Dorris Neurological Research Institute, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037, USA
Email: Henning Lauterbach - ; Phi Truong - ; Dorian B McGavern* -
* Corresponding author
Abstract
Once a virus infection establishes persistence in the central nervous system (CNS), it is especially
difficult to eliminate from this specialized compartment. Therefore, it is of the utmost importance
to fully understand scenarios during which a persisting virus is ultimately purged from the CNS by
the adaptive immune system. Such a scenario can be found following infection of adult mice with
an immunosuppressive variant of lymphocytic choriomeningitis virus (LCMV) referred to as clone
13. In this study we demonstrate that following intravenous inoculation, clone 13 rapidly infected
peripheral tissues within one week, but more slowly inundated the entire brain parenchyma over
the course of a month. During the establishment of persistence, we observed that genetically

Virology Journal 2007, 4:53 doi:10.1186/1743-422X-4-53
Received: 13 April 2007
Accepted: 6 June 2007
This article is available from: />© 2007 Lauterbach et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( />),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Virology Journal 2007, 4:53 />Page 2 of 16
(page number not for citation purposes)
(BBB) and the limited expression of antigen-presenting
machinery (i.e., major histocompatibility complex class I
and II) (reviewed in [5,6]). The downside of this tight
immune regulation is that a multitude of pathogens can
exploit this weakness in order to establish long term per-
sistence in CNS resident cells. Because the CNS is fraught
with mechanisms to limit the toxicity (and most likely the
effectiveness) of the immune response, it is surmised that
this tissue compartment provides a favorable environ-
ment for prolonged viral persistence and neurologic dys-
function long after sterilizing immunity is achieved in the
periphery (i.e., the route through which neurotropic
viruses enter naturally).
Fetal infection in humans with lymphocytic choriomen-
ingitis virus (LCMV) can lead to serious neurological com-
plications, such as microcephaly, hydrocephalus, reduced
mitosis in developing brain cells and mental retardation
[7]. If mice are infected at birth or in utero with LCMV,
neurons are the predominant cell population in the CNS
parenchyma that harbor the virus [8]. Intravenous infec-
tion of adult mice with the parental strain of LCMV
referred to as Armstrong results in an acute infection,

dated the brain parenchyma with delayed kinetics when
compared to peripheral tissues. Within the CNS paren-
chyma clone 13 sought early refuge within astrocytes and
later infected olfactory bulb neurons before it was eventu-
ally purged from the entire compartment. When the func-
tionality of the infiltrating CTL response was examined
over this protracted clearance phase, signs of CTL exhaus-
tion were evident but never as severe as that observed in
peripheral tissues such as the spleen and liver. Interest-
ingly, during the "functional reanimation" phase, a time
period when the anti-viral CTL response regained func-
tionality in all tissues, a major shift in the composition of
the CNS immune repertoire was observed. Most notably,
CD4 T and B cells increased both in frequency and cell
number within the CNS during this phase. This coincided
with a dramatic reduction in the number of persistently
infected astrocytes and the eventual eradication of clone
13 from the CNS. These data provide a framework for
understanding the cellular constituents responsible for
purging an established persistent infection from the CNS
and should facilitate future studies that aim to identify the
precise mechanism(s) of clearance.
Methods
Mice
C57BL/6 (H-2
b
, Thy1.2
+
) and C57BL/6 Thy1.1
+

PCR was performed on the cDNA product with primers
specific for the LCMV GP resulting in a 362 bp long DNA
fragment. 10 μg of the PCR product were digested with
Mnl I (NEB) and analyzed by agarose gel electrophoresis.
Virology Journal 2007, 4:53 />Page 3 of 16
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This method allows detection of the U-to-C change at
nucleotide 855 in the viral RNA of clone 13, which creates
a cleavage site for Mnl I.
T cell isolation and adoptive transfers
CD8 T cells were purified from the spleens of naïve P14
mice by negative selection (StemCell Technologies), and
5 × 10
3
purified cells were transferred i.v. into C57BL/6
mice. The mice were then infected 1–2 days later with
LCMV.
Mononuclear cell isolations and tissue processing
To obtain cell suspensions for flow cytometric analyses
and stimulation cultures, the spleens, livers and CNS were
harvested from mice after an intracardiac perfusion with a
0.9% saline solution to remove the contaminating blood
lymphocytes. If noted, organs were incubated with 1 ml
collagenase D (1 mg/ml; Roche) at 37°C for 20 min. Sin-
gle-cell suspensions were then prepared by mechanically
disrupting the organs through a 100-μm filter. Spleen cells
were treated with red blood cell lysis buffer (0.14 M
NH
4
Cl and 0.017 M Tris-HCl, pH 7.2), washed twice, and

min. The Fc block was also included in all 20 min surface
stains. For intracellular cytokine staining cell suspensions
were stimulated for 5 hrs with 5 μg/ml of a dominant CD8
epitope mapping to amino acids 33–41 of the LCMV glyc-
oprotein (GP
33–41
) in the presence of 50 U/ml recom-
binant IL-2 (NIH) and 1 μg/ml brefeldin A (Sigma).
Afterward, cells surface stained with CD8-Pacific Blue and
Thy1.1-PerCP and were then simultaneously fixed/perme-
abilized with a paraformaldehyde-saponin solution and,
finally, stained with antibodies directed against IFN-γ,
TNF-α and IL-2. Cells were acquired using a digital flow
cytometer (Digital LSR II; Becton Dickinson) that allows
up to 10-color detection by using four different excitation
lasers. Flow cytometric data were analyzed with FlowJo
software (Tree Star, Inc.). Gates for cytokine analyses were
set based on non-peptide-stimulated controls and cells
that stained negative for the protein of interest.
Immunohistochemistry
To visualize LCMV, astrocytes, and neurons, 6-μm frozen
sections were cut, fixed with 2% formaldehyde, blocked
with an avidin/biotin-blocking kit (Vector Laboratories),
and stained for 1 h at room temperature with guinea pig
anti-LCMV (1:1500), rabbit anti-glial fibrillary acidic pro-
tein (anti-GFAP; 1:800; DakoCytomation), or 1.25 μg/ml
of mouse anti-neuronal nuclei (anti-NeuN; Chemicon
International), respectively. To block endogenous mouse
antibodies, sections stained with mouse anti-NeuN were
pre-incubated for 1 hr at room temperature with 35 μg/ml

Distribution of LCMV in the brain and spleen following an intravenous clone 13 infection. Representative sagittal
brain and spleen reconstructions (n = 3 mice per group) were assembled at the denoted time points post-infection to reveal
the distribution of LCMV (green) following an intravenous infection with 2 × 10
6
PFU of clone 13. Note the minimal amount of
virus in the brain at day 10 and the complete inundation of the brain parenchyma by day 30. During the late phase of persist-
ence (day 150), clone 13 localizes primarily to the olfactory bulb (white arrow) and also maintains a presence in the meninges,
choroid plexus, ependyma, and subventricular zone. Note that the spleen shows the highest viral antigen load at day 10 and is
progressively purged of virus over time. Cell nuclei are shown in blue.
Virology Journal 2007, 4:53 />Page 5 of 16
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Clone 13 tropism in the brain parenchyma during persistenceFigure 2
Clone 13 tropism in the brain parenchyma during persistence. The localization of clone 13 in the brain parenchyma
was examined at various time points post-infection by two-color confocal microscopy. During the first 60 days the virus
(green) was found primarily in GFAP
+
astrocytes (red). Representative low (first row) and high (second row) magnification
images are shown for a mouse (n = 3 mice per group) at day 31 p.i. The third row shows a whole brain reconstruction from a
mouse (n = 3 mice per group) at day 150 and an enlarged panel of the olfactory bulb. Virus is shown in green and cell nuclei in
blue. In the late phase of persistence (day 150), the virus (green) was found primarily in NeuN
+
olfactory bulb neurons (red).
Low and high magnification examples are shown in the fourth and fifth rows, respectively.
Virology Journal 2007, 4:53 />Page 6 of 16
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LCMV-infected neurons or astrocytes (Fig. 2) were cap-
tured with a confocal microscope (MRC1024; Bio-Rad
Laboratories) fitted with a krypton/argon mixed gas laser
(excitation at 488, 568, and 647 nm) and a 40× oil objec-
tive (Carl Zeiss MicroImaging, Inc.). All two-dimensional

trast to the spleen (Fig. 1) and the liver (data not shown),
where antigenic load peaked at day 10 post infection
(p.i.), the brain parenchyma was not fully inundated with
clone 13 until day 30 (Fig. 1). Titers of infectious virus in
the CNS as measured by plaque assay reached their maxi-
mum level by day 20 p.i., and this titer was maintained
until day 60, at which point a steady decline in viral titers
was noted both by plaque assay (Table 1) as well as
immunohistochemistry (Fig. 1).
Interestingly, and in support of previous studies [20], the
pattern of clearance in the CNS did not closely mirror that
of peripheral tissues such as the spleen and liver. Whereas
the blood (data not shown), liver (data not shown), and
spleen (Fig. 1) were completely purged of virus within 60–
80 days of infection, CNS virus was not finally resolved
until around day 200 (Fig. 1, Table 1). However, coinci-
dent with the clearance of clone 13 from the periphery
around day 60 was a marked shift in the distribution of
virus within the brain parenchyma. Between day 60 and
150, clone 13 was purged to a large degree from the brain
parenchyma. In fact, the choroid plexus, meninges, sub-
ventricular zone, and, most notably, the olfactory bulb,
served as the last bastions of virus (see day 150, Fig. 1)
before the pathogen was finally purged at day 200 (Fig. 1).
These data demonstrate that despite the establishment of
long term persistence within the CNS, clone 13 can ulti-
mately be eliminated from this compartment; however,
the kinetics of clearance differ significantly from most
peripheral tissues.
Pattern and tropism of LCMV clone 13 in the CNS

brain parenchyma, but could still be found in the choroid
plexus, meninges, subventricular zone, and olfactory bulb
(Fig. 1). Interestingly, at this late phase of persistence,
clone 13 appeared to have acquired a new target. Co-stain-
ing analyses revealed that in addition to ependymal cells,
meningeal cells, and cells comprising the choroid plexus,
clone 13 had spread to olfactory bulb neurons (Fig. 2).
These data demonstrate that for the first two months of
persistence, clone 13 primarily infects astrocytes within
the brain parenchyma, but establishes late phase persist-
Virology Journal 2007, 4:53 />Page 7 of 16
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ence in olfactory bulb neurons before it is finally cleared
at day 200 post-infection.
Neurotropic Armstrong is not selected for over time in the
CNS of clone 13 infected mice
The localization of LCMV in olfactory bulb neurons dur-
ing the late phase of persistence suggested that the CNS
selected for the more neurotropic strain of LCMV (i.e.,
Armstrong) over time. There is precedence in the literature
to support that Armstrong can out-compete clone 13
when both are simultaneously administered into the CNS
[27]. Moreover, examination of viral clones extracted
from the CNS of LCMV carrier mice persistently infected
from birth has revealed that Armstrong is usually found in
the CNS and clone 13 in peripheral lymphoid tissues [11].
To determine if Armstrong was selected for in the CNS of
clone 13 infected mice over time, we examined viral
clones of LCMV extracted from the CNS at an early (day 8)
versus a late time point (day 150) p.i. The glycoprotein of

the endogenous CTL response [29-31]. The advantage of
using TCR-tg cells is that the fate of a single LCMV-specific
T cell population with a known TCR can be followed from
the initial infection to the late phase of persistence with-
Neurotropic LCMV Armstrong is not selected for in the CNS of clone 13 infected miceFigure 3
Neurotropic LCMV Armstrong is not selected for in the CNS of clone 13 infected mice. RNA was isolated from
LCMV clones extracted from the brains of mice at 21 (n = 7 clones) and 150 days (n = 6 clones) post-clone 13 infection. RT-
PCR, PCR and Mnl I restriction enzyme digests were performed as described in the Materials and Methods. The RNA PCR
product from the Armstrong GP contains a phenylalanine at position 260 and is not cleaved by Mnl I. In contrast, clone 13 con-
tains a leucine at position 260, and the 362 bp PCR product is cleaved into fragments (202 and 160 bp) by Mnl I. Note that all
clones analyzed at both time points had the Mnl I restriction enzyme site. The control lane shows undigested 362 bp GP PCR
product.
Table 1: Brain Viral Titers. Kinetics of viral clearance from the
brain. Clone 13 infected mice were perfused with saline and then
brains were isolated at the denoted days post infection (DPI).
The titer of infectious virus was determined by plaque assay and
is expressed as plaque forming units (PFU) per gram tissue. The
lower limit of detection is 200 PFU/g of tissue
DPI Brain Virus Titer (PFU/g)
10 4.04 × 10
5
20 3.90 × 10
6
30 2.16 × 10
6
60 1.16 × 10
5
150 5.73 × 10
3
200 < 200

(Fig. 4C,D). The marginal differences noted in the spleen
and liver did not reach statistical significance. During the
contraction phase following day 10 p.i., P14 cell numbers
remained elevated in the spleen and liver of clone 13
infected mice, but were eventually reduced to a steady
state level comparable to that observed in Armstrong
infected mice within one month of infection (Fig. 4A,B).
This steady state level was then maintained for the entire
examination period (200 days). Interestingly, at around
day 70 post-infection, a statistically significant (p = 0.016)
16-fold increase in the absolute number of P14 cells was
observed in the CNS (Fig. 4C,D), but not the spleen or
liver (Fig. 4A,B) of clone 13 infected mice when compared
to Armstrong. This increase coincided temporarily with
the decline in virus observed by both plaque assay (Table
1 and Fig. 4E) and immunohistochemistry (Fig. 1). It is
also worth noting that P14 cells were maintained in the
CNS of Armstrong infected mice for the entire observation
period despite our inability to detect virus at any time
point following day 10, supporting the notion that mem-
ory CTL are maintained in the CNS in the absence of anti-
gen [34,35]. Nevertheless, the marked increase of P14
cells observed in clone 13 infected mice suggests an anti-
gen-driven process.
Differential preservation of CTL function in clone 13
infected mice
One hallmark of chronic infection with clone 13 is the
gradual functional impairment of LCMV specific CD8
+
and CD4

the ability to produce cytokines in response to antigen
(Fig. 5A, day 60), and by day 90 P14 functionality was
fully restored in all tissues examined (Fig. 5A, day 90).
These data show that in the clone 13 system, CTL exhaus-
tion is followed by a period of "functional reanimation".
In addition, the severity of CTL exhaustion in the CNS was
never as great as that observed in peripheral tissues.
The "functional reanimation" phase is associated with
diversification in the CNS immune repertoire
The time course of sagittal brain reconstructions revealed
that clone 13 established widespread infection of the
brain parenchyma predominantly in astrocytes and that
the virus was finally eliminated from this compartment
following a transient state persistence in olfactory bulb
neurons (Fig. 1, 2). We define the time period following
day 60 as the "functional reanimation" phase because CTL
cytokine-producing ability returns to normal levels both
in the periphery and CNS. During this period, viral titers
in serum (not shown), liver (not shown), and spleen (Fig.
1) are reduced to background levels, and CNS virus begins
a steady descent that requires >100 additional days before
complete clearance is achieved. We became particularly
interested in this time period because the adaptive
immune system, despite passing through a state of func-
tional exhaustion, ultimately gains the upper hand in the
clone 13 system and purges virus from the immunologi-
cally specialized CNS. Therefore, we next examined the
immunological factors associated with CNS viral clear-
ance. Our CTL functional data demonstrate quite clearly
that immune exhaustion in the CNS was never as severe as

Thy1.1
+
P14 cells (open circles) in the CNS of clone 13 infected animals (as shown in panel C) is plotted against the titer
of infectious virus (black circles) in the brain at various time points after clone 13 infection (as shown in Table 1). Note that the
elevation in CNS CTL numbers coincides with a reduction in infectious virus as determined by plaque assay.
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Virology Journal 2007, 4:53 />Page 10 of 16
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Analysis of CTL function during clone 13 persistenceFigure 5
Analysis of CTL function during clone 13 persistence. A) Mononuclear cells were extracted from the spleen, liver and
CNS of Armstrong (black bars) or clone 13 (white bars) infected mice (n = 3 mice per group) at the denoted time points. Fol-
lowing a 5 hr in vitro stimulation with GP
33–41
peptide, P14 cells were examined flow cytometrically for the production of IFN-γ
(top row), TNF-α (middle row) and IL-2 (lower row). Note that when compared to the P14 cells in the spleen and liver, an
intermediate state of P14 functional exhaustion was observed in the CNS. This was most prominent at day 20 p.i. P14 cells in
all compartments regained complete functionality by day 90 p.i. Each bar represents the mean ± SD. Statistical differences
between Armstrong and clone 13 infected mice are denoted by asterisks (p < 0.05). B) Representative dot plots used to gen-
erate the bar graphs in panel A are shown for CNS P14 cytokine production at day 20 p.i. This time point was selected to
show the relative preservation of CNS P14 function at a time point when functional exhaustion was most severe in the spleen
and liver. Dot plots are gated on CD45
+
CD8

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Virology Journal 2007, 4:53 />Page 11 of 16
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However, because semi-functional CTL were maintained
in the CNS throughout the immune exhaustion stage of
infection, a time period when CNS viral loads were rela-
tively high, we postulated that CTL alone might not be
responsible for the eventual clearance of virus from the
CNS. To address this possibility we quantified the cellular
composition of CNS infiltrate during the reanimation
phase in clone 13 infected mice not seeded with traceable
P14 cells. When the ratio of bulk CD8 to CD4 T cells was
calculated in the spleen, liver and CNS over a 200-day
time window (Fig. 6A), we noted that, at the peak of the
primary response (day 8 p.i.), the CNS-infiltrating T cell
response was strongly dominated by CTL; there were 23
times more CD8 than CD4 T cells in the CNS on average.
Interestingly, this CD8 dominance was unique to the
CNS, because the spleen and liver at day 8 showed ratios
of 4.7 and 6.8, respectively. Even after the contraction
phase, the CNS still harbored 10 times more CD8 than
CD4 T cells. However, at the start of the functional reani-
mation phase (day 60–70 p.i.), the ratio stabilized
between 4–5 and remained there for the duration of the
examination period.

hi
NK1.1
-
Thy1.2
-
CD19
+
), NK cells (CD45
hi
CD4
-
CD8
-
CD11b
-
NK1.1
+
), dendritic cells (CD45
hi
NK1.1
-
Thy1.2
-
CD11c
+
),
macrophages (CD45
hi
NK1.1
-

MHC
tetramer as described previously [21,37]. At day 70, 4.5%
± 2.6% of the CD4 T cells were specific for the immuno-
dominant GP
61–80
peptide presented in I-A
b
. CD8 T cells
still represented the most predominant leukocyte popula-
tion in the CNS at day 70, with their numbers increasing
4-fold from day 8. In addition, 11.7 ± 2.6% of the CD8
cells were determined to be GP
33–41
specific by D
b
GP
33–41
MHC tetramer staining. Importantly, all of the aforemen-
tioned changes were unique to the CNS at day 70 and not
noted in the spleen or liver (data not shown). These data
demonstrate collectively that at day 70 p.i., a time point
when peripheral tissues are largely devoid of virus, the
adaptive immune response in the CNS not only regains
functionality (Fig. 5) but also diversifies its cellular reper-
toire (Fig. 6). This coincides with a decline in CNS viral tit-
ers (Fig. 4E).
Discussion
The immunosuppressive variant of LCMV, clone 13, was
first isolated over two decades ago from spleens of persist-
ently infected carrier mice [12], and since that time infec-

At the outset of our studies, little was known about the
progression of clone 13 infection within the CNS – a com-
partment of particular interest given its immunologically
specialized status [6,25] and its unique susceptibility to
irreparable consequences during viral persistence [1]. In
this model it was known that most peripheral tissues were
Virology Journal 2007, 4:53 />Page 12 of 16
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Diversification in the CNS immune repertoire during the reanimation phaseFigure 6
Diversification in the CNS immune repertoire during the reanimation phase. A) The CD8 to CD4 T cell ratio was
calculated for spleen, liver, and CNS of clone 13 infected mice over time by dividing the absolute number of CD8 T cells
extracted from each tissue by the absolute number of CD4 T cells. Note that the ratio is highest in the CNS at early time
points post-infection. As the number of CD4 T cells increase in the CNS over time this ratio becomes similar to that observed
in peripheral tissues. B) Mononuclear cells were extracted from spleen (data not shown), liver (data not shown) and CNS of
clone 13 infected mice (n = 4 mice per group) at day 8 (exhaustion phase) or day 69 (reanimation phase) p.i. to define the
immune repertoire. Multi-parameter digital flow cytometry permitted analysis of the entire immune repertoire from a single
sample for each tissue. The frequencies of both innate and adaptive immune cells are represented as pie diagrams. Statistically
significant increases (p < 0.05) at day 69 are denoted by asterisks. C) The frequencies shown in panel B were used to calculate
the absolute number of the respective CNS cell populations in clone 13 infected mice. Note the statistically significant (p <
0.05) increase in the number of CD4 T cells and B cells in the CNS at day 69. The bars represent mean ± SD at each time
point. Asterisks denote a statistically significant difference between day 8 and day 69.
Virology Journal 2007, 4:53 />Page 13 of 16
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purged of clone 13 within 50 to 60 days, whereas the CNS
remained replete with infectious virus at this time [43],
suggesting the possibility that the adaptive immune sys-
tem might not be equipped to cleanse the CNS of a per-
sistent virus after progressing through an
immunosuppressive state. Therefore, we initiated a series
of studies to investigate the relationship between clone 13

data suggest collectively that temporal diversification of
the immune repertoire is nature's solution to the problem
of removing immunosuppressive clone 13 from the
murine CNS – a supposition that requires further experi-
mentation to prove definitively.
To gain insights into clone 13 infection kinetics, we
assembled temporal sequences of tissue reconstructions
(periphery versus brain) to illustrate the expression of
viral antigen over time. After an intravenous injection,
clone 13 distributes systemically [12,23,24]. Following
systemic distribution, the representative peripheral tissues
we examined (i.e., spleen and liver) were fully inundated
with virus by day 10, whereas complete infection of the
brain parenchyma was not achieved until day 30 (Fig. 1).
This delay is likely explained by the presence of a non-
fenestrated blood-brain-barrier (BBB) in the CNS, which
has an essential role in maintaining a highly regulated
microenvironment for the proper neuronal functioning.
The BBB is composed of astrocytic foot processes,
endothelial cells, and their associated basement mem-
branes [44]. Importantly, the receptor for LCMV clone 13,
α-dystroglycan [45], is highly expressed on the astrocyte
foot processes [46-48]. In fact, we propose that this
explains the early targeting of astrocytes by clone 13. We
also postulate that astrocytes likely serve as the portal for
clone 13 entry into the CNS following intravenous inocu-
lation. This is supported by our confocal analyses (Fig. 2)
and the punctate pattern of viral antigen expression we
observed around blood vessels on brain reconstructions at
day 20 p.i. (Fig. 1). Astrocytes have also been described as

not generated, it remains possible that other variants of
LCMV were selected for in the CNS of clone 13 infected
mice. Sequence analyses of clones are required to address
this possibility.
A second possibility to explain the transition to olfactory
bulb neurons during the late phase of persistence could be
the targeting of neural stem cells. Interestingly, lentiviral
vectors pseudotyped with LCMV (WE54)-GP have been
shown to transduce neural stem cells/progenitors in vivo
[54]. Studies have demonstrated that GFAP-expressing
type B astrocytes residing in the subventricular zone (SVZ)
are the in vivo precursors of newly generated neurons in
Virology Journal 2007, 4:53 />Page 14 of 16
(page number not for citation purposes)
the adult mammalian brain [55]. Type B astrocytes give
rise to rapidly dividing transit-amplifying cells, which fur-
ther develop into migratory neuroblasts (reviewed in
[56]). Forming tangential chains, these neuroblasts
migrate along the rostral migratory stream (RMS) from
the SVZ into the olfactory bulb, where they differentiate
into two kinds of inhibitory neurons [57]. Given that len-
tiviral vectors pseudotyped with LCMV GP have been
shown to target neural stem cells, it conceivable that clone
13 accesses olfactory bulb neurons in part through this
pathway. Studies are underway to address this hypothesis.
Because clone 13 persisted in the CNS of mice for roughly
6 months before eradication, we considered analyses of
the responding immune repertoire to be of great impor-
tance. We began by first examining a traceable representa-
tive of the virus-specific CTL response, namely P14 cells

clone 13 persistence in the CNS was considerably longer
than that observed in peripheral tissues containing heav-
ily exhausted T cells. Therefore, the degree of functional
exhaustion cannot be used to explain the pattern of per-
sistence in the CNS following an intravenous clone 13
infection.
One of the most important features of the clone 13 model
is the ability of dysfunctional antiviral CTL to regain their
cytokine-producing abilities starting around day 60 p.i.
This progresses to a state of complete functional recovery
in all tissues examined by day 90 p.i. (Fig. 5). During this
"reanimation phase" clone 13 is purged from most of the
periphery [19,20], and we noted a marked elevation in the
number of CTL in the CNS. Associated with CTL reactiva-
tion was a dramatic shift in the immune repertoire found
in the CNS, but not the periphery (Fig. 6). Most notably,
a substantial increase in CD4
+
T cells (which included the
dominant I-A
b
GP
61–80
specific response) and B cells were
observed in the CNS. B cells and antiviral antibodies have
been implicated in the control of certain strains of LCMV
[58,59] as well as the CNS-tropic mouse hepatitis virus
(JHMV) [60,61]. In addition, studies have shown that pas-
sive administration of anti-LCMV antibodies (IgG2a iso-
type) can partially protect mice from the fatal

D.B. McGavern), AI070967-01 (to D.B. McGavern), and MH062261-06
(pilot grant to D.B. McGavern), a grant from The Dana Foundation (to D.B.
McGavern), and a grant from The Ray Thomas Edwards Foundation (to
D.B. McGavern). H. Lauterbach is supported by a fellowship from Deutsche
Forschungsgemeinschaft (DFG).
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